The overall goal of the present project is to elucidate the mechanisms of differentiation of the functional properties of the embryonic heart cell membrane, and the effects of peptide hormones on the electrical parameters. During the past year we have shown that insulin binds to both 7- and 14-day heart cells with a curvilinear Scatchard plot, indicating that two classes of insulin receptors exist. One class consists of a small number of high-affinity binding sites (KA equals 5 nM; 600 receptors/cell); the other is a low-affinity, high capacity system (KA equals 0.026 nM; 9000 receptors/cell). Occupancy of the low-affinity receptors stimulates AIB transport. Binding of 125I-insulin to the high-affinity system was reduced 50% after culture of heart cell aggregates in 20 micrograms/ml insulin for 8-44 hrs, while binding to the low-affinity receptors was unchanged. In binding competition studies, the relative potencies of three peptide hormones insulin:proinsulin:MSA were (1:0.05:0.03). In contrast, the relative potencies for stimulating A-system transport were MSA:insulin:proinsulin (3:1:0.28). When aggregates of 14-day ventricle cells were washed thoroughly in buffer they had resting potentials of minus 63 mV. Insulin caused a hyperpolarization of these cells in a dose-dependent manner with a half-maximal response (9mV) at 1 x 10 to the minus 9th power M. This effect began 1-2 minutes after hormone addition, and occurred even in the presence of 10-50 micromoles ouabain. Steady state current clamp I-V curves indicate that the insulin-induced hyperpolarization results from an increase in conductance to an ion whose equilibrium potential is more negative than minus 90 mV. This electrical effect of insulin was associated with occupancy of the high-affinity receptor system.